Terpenoids or terpenes represent a family of natural products found in most organisms (bacteria, fungi, animal, plants). Terpenoids are made up of five carbon units called isoprene units. They can be classified by the number of isoprene units present in their structure: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (Cn, n≧45). The plant kingdom contains the highest diversity of monoterpenes and sesquiterpenes.
The monoterpenes and sesquiterpenes are the most structurally diverse isoprenoids. They are usually volatile compounds and are mostly found in plants were they play a role in defense against pathogens and herbivores attacks, in pollinator attraction and in plant-plant communication.
Some plants, known as aromatic plants or essential-oil-plants, accumulate large amounts of monoterpenes and sesquiterpenes in their leaves, roots or stems. Classical examples of such plants are members from the plant families Lamiaceae, Rutaceae, Solanaceae, and Poaceae, for example.
Monoterpene and sesquiterpene accumulating plants have been of interest for thousands of years because of their flavor and fragrance properties and their cosmetic, medicinal and anti-microbial effects. The terpenes accumulated in the plants can be extracted by different means such as steam distillation that produces the so-called essential oil containing the concentrated terpenes. Such natural plant extracts are important components for the flavor and perfumery industry.
Many sesquiterpene compounds are used in perfumery. For example, Vetiver oil, extracted from the roots of Vetiver zizanoides, is known to contain a number of odorant sesquiterpenes, amongst which α-vetivone, β-vetivone and zizanoic acid, are the most characteristic. Vetiver zizanoides is currently cultivated in Reunion, the Philippines, Comoro Islands, Japan, West Africa and South America.
Generally, the price and availability of plant natural extracts such as Vetiver oil is dependent on the abundance, the oil yield and the geographical origin of the plants. In some years, the availability of commercially available natural extracts decreases, going hand in hand with a worsening of their quality. Under these circumstances, the use of these ingredients in high quality perfumery products is no longer possible.
Therefore, it would be an advantage to provide a source of sesquiterpenes, which is less subjected to fluctuations in availability and quality. Chemical synthesis would seem to be an evident option for the preparation of sesquiterpenes, however, these compounds generally have a highly complex structure and so far no economic synthetic process for the preparation of sesquiterpenes has been developed.
It is therefore an objective of the present invention to provide ways of producing a high quality of sesquiterpenes in an economic and reliable way.
The biosynthesis of terpenes in plants has been extensively studied and is not further detailed in here, but reference is made to Dewick P, Nat. Prod. Rep., 2002, 19, 181-222, which reviews the state of the art of terpene biosynthetic pathways.
The sesquiterpene synthases convert FPP to the different sesquiterpene skeletons. Over 300 sesquiterpene hydrocarbons and 3000 sesquiterpenoids have been identified (Joulain, D., and König, W. A. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998; Connolly, J. D., Hill R. A. Dictionary of Terpenoids, Vol 1, Chapman and Hall (publisher), 1991), and many new structures are identified each year. There is virtually an infinity of sesquiterpene synthases present in the plant kingdom, all using the same substrate but having different product profiles.
A cDNA encoding a trans-α-bisabolene synthase has been reported by Bohlmann, J, Crock, J., Jetter, R., and Croteau R. (1998) Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis). Proc. Natl. Acad. Sci. USA 95, 6756-6761. However, this enzyme catalyses one cyclation step and produces almost exclusively the bisabolene sesquiterpene.
Köllner T et al (2004) The plant cell 16(5), 1115-1131, disclose a number of putative terpene synthase genes isolated from Zea mays, most of which did not encode functional enzymes. DNA sequences of functional synthases, Tps4-B73 and Tps5-1 delprim are available under accession number AY518310 and AY518313. Bergamotene was only a minor product produced by the encoded enzymes, representing maximally 2.6 wt. % of the total sesquiterpenes produced.
Despite extensive chemical studies of terpene cyclisation, the isolation of the enzymes is difficult, particularly in plants, due to their low abundance, often transient expression patterns, and complexity of purifying them from the mixtures of resins and phenolic compounds in tissues where they are expressed.
In view of the above, the objective of the present invention is to provide new terpene synthases. Another objective is to isolate terpene synthases from the plant Vetiveria zizanoides. It is an objective of the present invention to provide terpene syntases capable of synthetizing terpenes for the synthesis of which so far no enzyme has been reported.
In particular, it is an objective to provide enzymes capable of synthesising substantial amounts of sesquiterpenes having a santalene or bergamotene carbon skeleton. There is no report of the genetic basis underlying a santalene synthase, and bergamotane is synthesised only in trace amounts by known terpene synthases.
In the same line, it is an objective to provide methods for making terpenoids in an economic way, as indicated above. Accordingly, the present invention has the objective to produce sesquiterpenes while having little waste, a more energy and resource efficient process and while reducing dependency on fossil fuels. It is a further objective to provide enzymes capable of synthesizing terpenoids, which are useful as perfumery and/or aroma ingredients.